Vitamin D (VD) is a vital substance for human survival that plays an important role in calcium and phosphorus absorption and bone mobilization. Vitamin D is either produced in human skin in a form known as Vitamin D3 (cholecalciferol, VD3) or it is absorbed from the diet, in a form known as Vitamin D2 (ergocalciferol, VD2). Both VD3 and VD2 undergo activation by their hydroxalation in the liver (25OHVD3 and 25OHVD2) and are further metabolized by additional hydroxylation in the kidneys to 1,25(OH)2VD3, 24,25(OH)2VD3, 1,25(OH)2VD2, and 24,25(OH)2VD2,
Typically, measurement of Vitamin D in humans is performed by measurement of VD metabolites rather than the inactive VD prescursors. Metabolites 25OHVD3, 1,25(OH)2VD3, 24,25(OH)2VD3, 25OHVD, 1,25(OH)2VD2, and 24,25(OH)2VD2 and other VD metabolites are generally referred to herein as “Vitamin D or VD.”
Since Vitamin D levels in humans are directly affected by the function of the kidneys and liver, and since Vitamin D levels directly affect the regulation of calcium and phosphorous, the ability to measure Vitamin D in blood is important to the diagnosis and study of a broad range of diseases, including diseases of the bones, kidneys, and liver. Many other analytes in the broad category of secosteroids are similarly important for diagnostic purposes.
In the past, blood levels of secosteroids, including, for example, Vitamin D, have been determined from biological samples by methods including high-performance liquid chromatography, mass spectrometry, competitive protein binding assays, or other quantification techniques such as enzyme assays, immunoassays, chemical colorimetric assays, or fluorescence labeling. Methods known to the art further include methods of derivatizing analytes using Cookson-type reagents such as PTAD to generate a derivatized secosteroid or Vitamin D metabolites, purifying or extracting these analytes using liquid chromatography, and analyzing the purified sample for quantities or concentrations of the analytes using mass spectrometry. Current vitamin D analysis methods often lack the sensitivity and specificity required to address analytical problems pressing to the art, particularly attempts to discern the tissue distribution of the many forms of vitamin D. Commercially available kit assays known to the art allow high throughput analysis of 25(OH)D, but not of vitamin D, and inter-laboratory performance of these kits is poor. Kits known to the art generally utilize an extraction method from serum based on acetonitrile, followed by column separation to separate 25(OH)D from other metabolites. Kits using this method are unable to accurately and separately measure 25(OH)D3 and 25(OH)D2.
Also known to the art is the use of high-performance liquid chromatography (HPLC) coupled with mass spectrometry (LC-MS). This method offers increased sensitivity and selectivity over kits, particularly when Atmospheric pressure chemical ionization (APCI) is employed in combination with a Multiple Reaction Monitoring (MRM) technique. LC-MS techniques known to the art relying on APCI, however, also suffer several deficiencies. In particular, APCI often leads to the premature fragmentation of vitamin D molecules during ionization, decreasing the quality of and sensitivity of analysis and contributing to a higher variability with high LOQs.
Methods known to the art are further unable to establish satisfactory ionization efficiencies for the low (fmole) levels of some secosteroids analytes expected in biological matrices. Although attempts have been made to use electrospray ionization (ESI) in LC-MS to address this issue, the effectiveness of ESI is analyte-dependent and the structure of Vitamin D related molecules suggests they would not readily protonate during ESI, resulting in poor, if any, detection.
Methods of determining secosteroid levels in biological samples currently known to the art suffer a number of further disadvantages, including the length of time needed to complete the assay, level of accuracy, sensitivity, and cost. For instance, one approach for the extraction of Vitamin D and its isoforms common to the art is to deproteinize a sample solution suspected to contain Vitamin D, such deproteinization adapted to release Vitamin D metabolites that are bound to proteins within the sample solution. Such released Vitamin D may then be extracted using an organic solvent or derivatized using a Cookson-type reagent. Performing a liquid:liquid extraction and/or derivatization process of this kind typically is lengthy, and it requires a relatively large volume of available sample solution to generate a sufficient volume of extracted Vitamin D to analyze using chromatographic and/or MS separation and analysis techniques. Such traditional methods of performing assays are further labor intensive, requiring personnel to manually complete a series of preliminary tasks such as extraction, centrifugation, evaporation, and derivatization before the sample can be separated or purified, and, finally, analyzed for levels of the desired analytes. Further, derivatization with Cookson-type reagents is itself a comparatively lengthy process, routinely taking several hours to complete.
Accordingly, it is an object of versions of the present invention to provide a method of assaying biological samples for the levels, amounts, or concentrations of analytes, including specifically secosteroids, and most specifically Vitamin D, with improved accuracy and sensitivity, and without the need for lengthy and labor-intensive preparation steps prior to analysis. It is further an object of versions of the present invention to provide a method of assaying biological samples for Vitamin D with a reduced number of steps. These and other advantages are provided in the methods described below, and still further advantages to the methods claimed herein will be apparent to one skilled in the art.